Neovascularization is an essential developmental process that is activated under pathological conditions and controlled by the expression of growth-promoting and growth-suppressing angiogenesis factors (Beck & D'Amore, 1997; Ferrara, 2000; Folkman, 1995; Kerbel, 2000; Li, 2000). During the initial activation stage, tumor or epithelial cells secrete angiogenic factors such as VEGF, FGF, and angiopoietins that alter cell cycle kinetics and stimulate EC proliferation or migration (Carmeliet & Collen, 2000; Hanahan, 1997; Maisonpierre et al., 1997). In later stages, tube formation and vessel maturation lead to vessel remodeling and apoptosis, which are regulated by TGFβ1 and mesenchymal cells in the absence of EC proliferation (Beck & D'Amore, 1997; Pepper et al., 1990; Taipale & Keski-Oja, 1997). This complex process of gene expression is regulated by a variety of transcription factors whose functions in angiogenesis have been deduced from targeted gene disruption studies in vivo or in cultured cells (Sato, 2000).
The Runx genes are a conserved family of DNA binding proteins containing a unique Runt homology domain (RHD) originally described in Drosophila (Ito, 1999). The RUNX proteins are members of the Ig-loop DNA binding family of proteins that include Stat1, p53, and NFkB (Bravo et al., 2001). Runx proteins are phosphorylated (Selvamurugan et al., 2000; Xiao et al., 2000) and associate with the core-binding factor-β (Cbfβ) in the nucleus to bind a specific nucleotide sequence. Several key observations support a role for Runx genes in angiogenesis including the finding that mice in which either the Runx1 or Runx2 genes have been disrupted die in utero or soon after birth with vascular abnormalities (Li et al., 2002; Lund & Van Lohuizen, 2002). Runx1 negative mice fail to recruit hematopoiefic stem cells for angiogenesis and exhibit defective vessel formation in the pericardium and head (Takakura et al., 2000). In Runx2-deficient mice, there is no vascular or mesenchymal cell invasion in cartilage, no evidence of VEGF expression in hypertrophic chondrocytes, and consequently no bone formation (Komori et al., 1997; Otto et al., 1997; Zelzer et al., 2001). The absence of VEGF may be a direct consequence of reduced Runx2 binding to the VEGF promoter (Zelzer et al., 2001). Conversely, VEGF, along with several angiogenic factors including FGF-1 and IGF-1, stimulate RUNX2 expression and migration of EC in vitro and in vivo (Namba et al., 2000; Sun et al., 2001). Reports of RUNX1 expression in human vascular EC and brain tumor cells in vivo also indicate that Runx genes are upregulated in highly-vascularized malignant tumors (Perry et al., 2002).
RUNX2 contains two domains not shared by other Runx family proteins: a QA-rich domain important in regulating transcription and a domain of unknown function encoded in exon 8 (Westendorf & Hiebert, 1999). Alternatively-spliced Runx2 isoforms have been reported (Stewart et al., 1997), including alternatively spliced exon 8 (Zhang et al., 1997), which exhibited reduced transactivation relative to RUNX2 (Geoffroy a al., 1998), as well as isoforms arising from alternative transcriptional start sites (Xiao et al., 1998).